Multimodal Fluorescence Light Sheet Microscopy (mFLSM) for in toto imaging of embryonic development – MFLSM-intotomorph

An embryo is shaped by a complex sequence of cell processes and morphogenetic events that are highly regulated both in time and space. Many of these mechanisms, such as collective cell migration, tissue invagination, or cell intercalation, are stereotypical and occur in every organisms. Advances in genetics or in molecular and cell biology, provided insights and tools to improve our understanding of the mechanisms involved in embryonic development. However, most of the studies in developmental biology are limited to the scale of a few cells or to the scale of the entire organism without cellular resolution. In addition, the challenging experimental conditions often limit the investigation to static analysis using fixed embryos. Hence, fundamental mechanisms involving the dynamic interaction of large group of cells are poorly understood. These mechanisms involved cell and molecular events or cell-cell interactions that are highly dynamics and have consequences at the scale of the entire organism. For these reasons, their study requires an experimental approach that is in vivo, dynamic, and multiscale, from the cell level to the scale of the organism. In parallel, recent advances in microscopy and in image processing, such as multiphoton microscopy, Fluorescence Light Sheet Microscopy, or 3D-cell tracking, enable to investigate the dynamics of large cell populations. These advances lead to the emergence of the in toto imaging concept and provides a unique opportunity to study embryonic development from the scale of the cellular mechanisms to the entire organism. in toto imaging of embryonic morphogenesis aims at imaging an entire organ or organism with cellular resolution during its development and at analyzing the digital data in a standard and quantitative manner. This promising approach involves several experimental challenges including the in vivo imaging of an entire tissue or embryo in a manner that does not compromise normal biology and viability. In addition, the complexity of the multidimensional data generated warrants the use of sophisticated quantitative analyses.
In this context, the purpose of this proposal is to bring novel approaches and techniques of in toto imaging to unravel fundamental processes of early embryonic morphogenesis. This project will be conducted at Institut Jacques Monod (IJM, Paris) and will gather an interdisciplinary group of scientists. Willy Supatto, the principal investigator, will use his training and experience in optics, biophysics and developmental biology to start a new research activity within Jérôme Collignon’s team at IJM. This team is studying cellular fate during mouse embryonic development. The project also involved a collaboration with Julien Vermot’s team at Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC, Illkirch, France), whose research is focused on the roles of biological flows during zebrafish embryogenesis. Different cutting edge approaches will be used, including the development and characterization of 2-photon excited Fluorescence Light Sheet Microscopy (2pFLSM), a new technique with improved depth penetration and acquisition speed compared to current techniques, which makes it particularly adapted for in toto imaging of embryos. A systematic image analysis approach will be developed to analyze the data generated and quantify stereotypic morphogenetic events. These methodological innovations will be developed using three different model systems (Dosophila, zebrafish and mouse embryos) and applied to the study of axis specification and symmetry breaking in early vertebrate embryos.